The goal of this proposal is to construct, optimize, and test a bench top x-ray fluorescence computed tomography (XFCT) system based on a promising new geometry we have recently developed and validated using synchrotron radiation. The novel geometry involves pencil-beam x-ray illumination of the sample coupled with slit collimation of a position- and energy-sensitive fluorescence x-ray detector. This allows for direct acquisition of the distribution of elements along the illuminated line without solving an ill-posed inverse problem. While the technology has the potential to be used in vivo, our aim in this proposal is to perform very high quality ex-vivo imaging of trace metals in biological samples. Many endogenous metals and metal ions, such as iron, copper, and zinc, play critical roles in signal transduction and reaction catalysis, while others, such as mercury, cadmium, and lead, are quite toxic even in trace quantities. In the post-genomic era, the new disciplines of metallogenomics, metalloproteomics, and metallomics are emerging for the systematic study of endogenous metals. These disciplines would benefit greatly from the spatially resolved maps of trace-element distribution and speciation provided by the methods being explored in the proposal. We seek to construct a system that can image sub-centimeter specimens (such as mouse organs) at 100 micron spatial resolution, with reasonable imaging times (~ 1-4 hours) and at radiation doses below damage threshold. The specific aims of the proposal are: Aim 1: The system design will be optimized for sensitivity using analytic and Monte Carlo-based tools Aim 2: A benchtop XFCT system will be fabricated Aim 3: Calibration procedures and image formation algorithms will be developed Aim 4: The system will be tested on phantoms and samples of biological interest